The Continuing Challenge of Carbapenemases in K. pneumoniae: KPC-2 & NDM-1

描述(由申请人提供)： 碳青霉烯耐药革兰氏阴性病原体的出现和传播是与医疗保健相关的发病率和死亡率的重要来源。碳青霉烯酶(即碳青霉烯类水解酶)有可能灭活所有的内酰胺类抗生素，严重限制了可用于治疗这些细菌感染的抗菌药的使用。目前，克雷伯菌中出现了两种主要的碳青霉烯酶：肺炎克雷伯菌碳青霉烯酶(KPC-2)和新德里金属内酰胺酶(NDM-1 MBL)。KPC是一种A类碳青霉烯酶，可水解亚胺培南、厄他培南、美罗培南、多利培南和内酰胺酶抑制剂，在美国几乎每一家主要的三级医疗机构中都有发现。为了设计未来的内酰胺类药物来对抗 为了应对这些迫在眉睫的威胁，我们需要了解拥有KPC和NDM-1的菌株的底物分布和敏感性模式将如何由于β-内酰胺酶的“热点”(即&-环，KPC-2中的氨基酸残基164到179，以及NDM-1的活性部位的环区和入口)的替换而发生变化。这些挑战构成了将在本文中讨论的研究方向。在这个优点建议中，我们将提出的主要研究问题是：1)如果在KPC-2中出现&环中的替换，它们是否会通过呈现将给临床医生带来更大挑战的新表型而构成显著的临床威胁？2)NDM-1 MBL的独特结构特征如何影响内酰胺酶降解内酰胺类抗生素的能力？为了回答第一个问题，我们的计划是确定KPC-2中&-环氨基酸位置R164、E166、N170和D179的替换的影响，并确定这些突变如何改变微生物表型、蛋白质稳定性和催化机制。为了解决第二个问题，我们将使用突变技术逐步去除NDM-1中163到166(163-166)位氨基酸的“独特的附加序列”，并评估这些缺失对耐药性(表型)、标准内酰胺类和碳青霉烯类底物的水解以及蛋白质稳定性的影响。接下来，我们将使用突变来取代(位置饱和)并删除NDM-1中的F70(70)，NDM-1是活性位点入口处的一种氨基酸，并评估该变异体的活性及其在蛋白质稳定性和催化中的作用。通过关注肺炎克雷伯菌的碳青霉烯酶，我们将提供导致新疗法的关键知识。我们的实验室正在积极确定KPC-2的氨基酸序列要求，这一新项目将作为进一步探索&LOOP作用和NDM-1生化研究的重要任务而脱颖而出。实现我们的目标将促进药物化学家设计有效的治疗剂所需的一般理解，这些治疗剂将作为这些“多功能”内酰胺酶的底物和抑制剂，并帮助我们设计抗生素治疗方案，以帮助医生面对这种严重和新出现的传染病威胁。

DESCRIPTION (provided by applicant): The emergence and dissemination of carbapenem-resistant Gram-negative pathogens is a significant source of healthcare-associated morbidity and mortality. Carbapenemases (i.e., carbapenem-hydrolyzing ¿-lactamases) that potentially inactivate all ¿-lactams severely limit the antimicrobials available to treat infections with these bacteria. Presently, two major carbapenemases are emerging in Klebsiella and are a major threat to our antibiotic armamentarium: the Klebsiella pneumoniae carbapenemase (KPC-2), and the New Delhi Metallo-¿-lactamase (NDM-1 MBL). KPC is a class A carbapenemase that hydrolyzes imipenem, ertapenem, meropenem, doripenem, and ¿- lactamase inhibitors and is found in almost every major tertiary care facility in the US. In order to design future ¿-lactams to combat these imminent threats, we need to understand how the substrate profile and susceptibility patterns of strains that possess KPC and NDM-1 will change as a result of substitutions in "hot spots" in the ¿-lactamase (i.e., &-loop, amino acid residues 164 to 179 in KPC-2, and the loop regions and entrance to the active site of NDM-1). These challenges frame the research direction that will be addressed herein. The major research questions we will ask in this Merit Proposal are: 1) If substitutions in the &-loop arise in KPC-2, will they pose a significant clincal threat by representing novel phenotypes that will present even greater challenges to the clinician? 2) How do unique structural features of NDM-1 MBL impact the ability of the ¿-lactamase to hydrolyze ¿-lactams? To answer the first question our plan is to determine the effect of substitutions in the &-loop at amino acid positions R164, E166, N170, and D179 in KPC-2 and ascertain how these mutations alter microbiological phenotype, protein stability and catalytic mechanism. To address the second question we will use mutagenesis to remove, in a stepwise manner, the "distinctive additional sequence" at amino acid positions 163 to 166 ( 163-166) in NDM-1 and assess the impact of these deletions on resistance (phenotype), hydrolysis of standard ¿-lactam and carbapenem substrates, and protein stability. Next, we will use mutagenesis to replace (site-saturation) and to delete F70 ( 70) in NDM-1, an amino acid at the entrance of the active site and assess the activity of this variant and its role in protein stability and catalysis. By focusing on the carbapenemases of K. pneumoniae we will provide the critical knowledge that leads to novel therapies. Our laboratory is actively defining the amino acid sequence requirements of KPC-2 and this new project will stand out as an important undertaking in the further exploration of the role of the &-loop and in the biochemical study of NDM-1. Achieving our objectives will advance the general understanding needed for the medicinal chemist to design effective therapeutic agents that will act as substrates and inhibitors against these "versatile" ¿-lactamases and help us devise antibiotic treatment regimens that will assist physicians faced with this serious and emerging infectious disease threats.